The overall goal of these studies is to elucidate the factors that control bile acid synthesis and activity of cholesterol 7alpha-hydroxylase, the rate limiting enzyme in BA synthesis. We will test the effects of agents which may rapidly affect the synthesis of bile acids from 14C-cholesterol in freshly isolated rat hepatocytes and/or in monolayer cultures. Active agents will be examined for their effects on 7alpha-hydroxylase by measuring its activity in microsomes isolated from cells incubated in the presence and absence of the test agent. Based on our preliminary experiments in isolated hepatocytes and with 7alpha-hydroxylase in liver microsomes, we propose that this enzyme is activated by phosphorylation. Therefore, hepatocyte studies will initially focus on agents that may affect 7alpha-hydroxylase because they are know to alter the activity of protein kinases. Such agents include glucagon, alpha-adrenergic agents, vasopressin, angiotensin-II, Ca++ depletion, Ca++ ionophores, and chlorpromazine. In addition, we will extend our observations on the effects of phosphorylation-dephosphorylation on the activity of 7alpha-hydroxylase in isolated liver microsomes. We will investigate the effects of phosphatases and protein kinases on the activity of the enzyme in isolated rat liver microsomes and purify the cytochrome P-450 and NADPH cytochrome P-450 reductase components of 7alpha-hydroxylase to determine which component is affected by phosphorylation. Monolayer cultures will be used to study factors, such as insulin, thyroxine, estrogen, corticosteroids, free and conjugated bile acids, cholesterol, and 25-hydroxycholesterol that may regulate bile acid synthesis by changing cellular levels of 7alpha-hydroxylase. Finally, we will investigate the effects of in vivo perturbations, such as nutritional state and administration of cholestyramine, cholesterol, or mevalonolactone, on the activity and phosphorylation state of 7alpha-hydroxylase in isolated liver microsomes. Since bile acids are an important end product of cholesterol metabolism and are needed to help solubilize biliary cholesterol, studies on the regulation of bile acid synthesis have important implications for the prevention of atherosclerosis and gallstones.